The Moonlighting Function of Soybean Disordered Methyl-CpG-Binding Domain 10c Protein.

Intrinsically disordered proteins (IDPs) are multifunctional due to their ability to adopt different structures depending on the local conditions. The intrinsically disordered regions of methyl-CpG-binding domain (MBD) proteins play important roles in regulating growth and development by interpreting DNA methylation patterns. However, whether MBDs have a stress-protective function is far from clear. In this paper, soybean GmMBD10c protein, which contains an MBD and is conserved in Leguminosae, was predicted to be located in the nucleus. It was found to be partially disordered by bioinformatic prediction, circular dichroism and a nuclear magnetic resonance spectral analysis. The enzyme activity assay and SDS-PAGE results showed that GmMBD10c can protect lactate dehydrogenase and a broad range of other proteins from misfolding and aggregation induced by the freeze-thaw process and heat stress, respectively. Furthermore, overexpression of GmMBD10c enhanced the salt tolerance of Escherichia coli. These data validate that GmMBD10c is a moonlighting protein with multiple functions.

Functional characterization of an unknown soybean intrinsically disordered protein in vitro and in Escherichia coli.

Intrinsically disordered proteins (IDPs) possess a wide range of biological function in all organisms, however the specific functions of most IDPs are still unknown. Soybean LOC protein, LOC for short, is a heat-stable protein, which is more abundant in the stress-resistant radicles. Sequence alignment and phylogenetic analysis showed that LOC is a functionally unknown protein and conserved in Fabaceae. LOC, being enriched in most disorder-promoting residues and depleted in most order-promoting residues, was predicted to contain high levels of intrinsic disorder by several commonly used computational tools. However, it was also predicted to contain two disorder-based protein-protein binding sites and two short α-helical segments. The circular dichroism spectroscopic analysis showed that this protein is mostly disordered in water, but can form more α-helical structure in the presence of SDS and TFE. Functional in vitro studies showed that the LOC protein is able to prevent lactate dehydrogenase inactivation by freeze-thaw at a molar ratio of 10:1. Furthermore, in vivo analyses revealed the survival rate of Escherichia coli over-expressing LOC protein under the conditions of osmotic stress was noticeably increased in comparison with the control. These observations suggest that the intrinsically disordered protein LOC might serve as a chaperone and/or cell protector.

Selection of mutant µplasmin for amyloid-ß cleavage in vivo.

One of the main culprits of Alzheimer's disease (AD) is the formation of toxic amyloid-ß (Aß) peptide polymers and the aggregation of Aß to form plaques in the brain. We have developed techniques to purify the catalytic domain of plasmin, micro-plasmin (µPlm), which can be used for an Aß-clearance based AD therapy. However, in serum, µPlm is irreversibly inhibited by its principal inhibitor α2-antiplasmin (α2-AP). In this study, we engineered and selected mutant forms of µPlm that are both catalytically active and insensitive to α2-AP inhibition. We identified surface residues of µPlm that might interact and bind α2-AP, and used an alanine-scanning mutagenesis method to select residues having higher activity but lower α2-AP inhibition. Then we employed saturation mutagenesis for further optimize both properties. Modeled complex structure of µPlm/α2-AP shows that F587 is a critical contact residue, which can be used as a starting position for further investigation.